1. Field of the Invention
The present invention relates to novel nucleoside and phosphonoacid lipophilic conjugates and methods of treatment such as treatment of virally infected cells by administering one or more of the conjugates of the invention. Compounds of the invention include phosphonoacid/nucleoside conjugates where the carboxyl group and phosphonyl groups of the phosphonoacid are esterified whereby the compound contains at least one lipophilic group and at least one nucleoside group.
2. Background
The human immunodeficiency virus type 1 (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV) and, to a lesser extent, human immunodeficiency virus type 2 (HIV-2) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. See, for example, Barre-Sinoussi et al., Science, 220:868-871 (1983); Gallo et al., Science, 224:500-503 (1984).
Methods for treating individuals infected by HIV have focussed on preventing integration of the virus into the host cell""s chromosome or on stages other than provirus. Thus one area of interest has been drugs that affect reverse transcriptase of HIV.
A number of dideoxy nucleosides have shown activity as reverse transcriptase inhibitors. In particular, AZT (zidovudine, 3xe2x80x2-azido-3xe2x80x2-deoxythymidine), ddI (2xe2x80x2,3xe2x80x2-dideoxyinosine), ddC (2xe2x80x2,3xe2x80x2-dideoxycytidine), d4T (2xe2x80x2,3xe2x80x2-dideoxy-2xe2x80x2,3xe2x80x2-dihydro-thymidine), (xe2x88x92) 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-thiacytosine (3TC), (xe2x88x92) 2xe2x80x2,3xe2x80x2-dideoxy-5-fluoro-3xe2x80x2-thiacytosine (FTC) and 1592U89 (Glaxo-Wellcome) have been used clinically for treatment of HIV infections.
Foscarnet, the trisodium salt of phosphonoformic acid (PFA, HOOCP(xe2x95x90O)(OH)2) is also a potent inhibitor of reverse transcriptase from human immunodeficiency virus type 1 (HIV-1). PFA inhibits replication of the virus in vitro and has been used clinically against AIDS. See E. Helgstrand et al., Science, 201:819-821 (1978); B. Oberg, Pharmacol. Ther., 40:213-285 (1989); H. Sundquist et al., J. Gen. Virol., 45:273-281 (1979); L. Vrang et al., Antimicrob. Agents Chemother., 29:8967-872 (1986); E. G. Sandstrom et al., Lancet, ii: 1480-1482 (1985); J. Gaub et al., AIDS Res., 1:27-33 (1987); M. A. Jacobson, J. Infect. Dis., 158:862-865 (1988); and C. V. Fletcher et al., Antimicrob. Agents Chemother., 38:604-607 (1994). PFA also inhibits DNA polymerase from cytomegalovirus (CMV), herpes simplex virus (HSV) and other DNA viruses, and PFA has been particularly useful in treating cytomegalovirus. See B. Eriksson et al., Biochim. Biophys. Acta, 607:53-64 (1980); C. S. Crumpacker, Am. J. Med., 92:3-7S (1992); O. Ringden et al., Lancet, i: 1502-1504 (1985); M. A. Jacobsen et al., Antimicrob. Agents Chemother., 33:736-741 (1989); A. G. Palestine et al., Ann. Intern. Med., 115:665-673 (1991); S. Safrin et al., N. Engl. J. Med., 325:551-555 (1991); and M. M. Reddy, J. Infect. Dis., 166:607-610 (1992). Phosphonoacetic acid (PAA, HOOCCH2P(xe2x95x90O)(OH)2) also exhibits antiviral activity. See U.S. Pat. No. 4,771,041 to Eriksson et al.
These known agents have well recognized limitations. For example, therapy with AZT (zidovudine), 3TC and other dideoxynucleosides has not prevented the breakdown of the immune system in many patients after a number of years of treatment. Still further, HIV strains have been reported that exhibit substantial resistance to AZT therapy and treatment with other known dideoxy nucleosides such as ddC, ddI, d4T and 3TC.
PFA does not have a high degree of oral absorption and consequently is generally administered intraveneously. PFA therapy also can result in toxicity to kidneys and hypocalcemia. Crisp et al., Drugs, 41:109-129 (1991).
Clinical resistance to PFA is known to occur after prolonged treatment. PFA resistant HIV-1 strains also have been produced in the laboratory by random as well as site-specific mutagenesis, and the pattern of cross resistance of such mutants to other reverse transcriptase inhibitors has been extensively analyzed. Mellors et al., Antimicrobial Agents and Chemotherapy, 39:1087-1092 (1995); Tachedjian et al., J. Virol., 70:7171-7181 (1996).
Additionally, the triple negative charge of PFA at physiological pH is an impediment to cellular uptake. As a result, the PFA concentration needed to block viral replication in an intact cell or in vivo is orders of magnitude greater than the concentration needed to inhibit the enzyme in a cell-free assay. Further, in vivo clearance of PFA is very rapid, which makes longlasting control of viral infection difficult to achieve.
Certain PFA derivatives have been reported, including certain simple alkyl and aryl esters of the carboxyl and/or phosphonyl moiety of PFA, certain acyloxymethyl esters of the phosphonyl moiety as well as certain ester derivatives in which the carboxyl or phosphonyl group was joined to a nucleoside. See J. O. Noren et al., J. Med. Chem., 26:264-270 (1983); L. R. Phillips et al., Tetrahedron Letters, 30:7141-7144 (1989); R. P. Iyer et al., J. Pharm. Sci, 83:1269-1273 (1994); M. Vaghefi et al., J. Med. Chem., 29:1389-1393 (1986); H. Griengl et al., J. Med. Chem., 31:1831-1839 (1988); A. Rosowsky et al., Biochem. Biophys. Res. Commun., 172:288-294 (1990); J. Sahaet al., Nucleosides and Nucleotides, 10:1465-1475 (1991); and A. S. Charvet et al., J. Med. Chem., 37:2216-2223 (1994). However, many of such compounds generally have not provided significant gains in terms of either potency or therapeutic selectivity for virally infected cells.
It thus would be desirable to have new compounds for treatment of virally infected cells, including cells infected with a retrovirus, particularly HIV. It would be especially desirable to have new compounds for treatment of cells infected with HIV strains that are resistant to current HIV therapeutics such as AZT and PFA.
We have discovered certain lipophilic phosphonoacid/nucleoside conjugates (covalently linked) that exhibit significant antiviral activity.
The invention thus provides methods of treatment against virus infections, including retroviral infections such as HIV infections, and treatment of other diseases caused by or otherwise associated with a virus such as influenza including influenza A and B; diseases associated with viruses of the herpes family, e.g., herpes simplex viruses (HSV) including herpes simplex 1 and 2 viruses (HSV 1, HSV 2), varicella zoster virus (VZV; shingles), human herpes virus 6, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and other herpes virus infections such as feline herpes virus infections; diseases associated with hepatitis viruses including hepatitis B viruses (HBV); and the like.
Particularly preferred compounds of the invention are active against drug-resistant viral strains. Indeed, it has been surprisingly found that compounds of the invention are highly active against HIV strains that are PFA-resistant as well as HIV strains that are AZT-resistant.
Compounds of the invention include phosphonoacid/nucleoside conjugates where the carboxyl group and phosphonyl groups of the phosphonacid are esterified whereby the compound contains at least one lipophilic group and at least one nucleoside group. More specifically, the invention provides compounds of the following Formula I that are highly useful to treat viral infections: 
wherein at least one of R, Rxe2x80x2 and Rxe2x80x3 is present, and at least one of N, Nxe2x80x2 and Nxe2x80x3 is present;
each R, Rxe2x80x2 or Rxe2x80x3 are each independently an optionally substituted alkyl having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkenyl having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkynyl having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkoxy having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkylthio having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkylsulfinyl having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; optionally substituted alkylsulfonyl having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons; or optionally substituted alkylamino having from about 8 to 30 carbon atoms, preferably about 14 to 24 carbons;
X and Y (if present where m is 1) are each independently hydrogen; halogen; hydroxyl; sulfhydryl; amino; optionally substituted alkyl preferably having 1 to about 12 carbons, more preferably 1 to about 6 carbons; optionally substituted alkenyl preferably having from about 2 to 12 carbon atoms, more preferably about 2 to 6 carbons; optionally substituted alkynyl preferably having from about 2 to 12 carbon atoms, more preferably about 14 to 24 carbon atoms; optionally substituted alkoxy preferably having 1 to about 12 carbon atoms, more preferably 1 to about 6 carbon atoms; optionally substituted alkylthio preferably having from about 1 to 12 carbon atoms, more preferably about 1 to 6 carbon atoms; optionally substituted alkylsulfinyl preferably having from about 1 to 12 carbon atoms, more preferably about 1 to 6 carbon atoms; optionally substituted alkylsulfonyl preferably having from about 1 to 12 carbon atoms, more preferably about 1 to 6 carbon atoms; or optionally substituted alkylamino preferably having from about 1 to 12 carbon atoms, more preferably about 1 to 6 carbon atoms;
W and Wxe2x80x2 are the same or different and each is independently O, S or Se;
m is an integer equal to 0 or 1;
each Z, Zxe2x80x2 and Zxe2x80x3 is independently hydrogen or a pharmaceutically acceptable cation such as a sodium, potassium, lithium, ammonium or quaternary ammonium (e.g. N(C1-6 alkyl)4+), and Z also may be optionally substituted alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl or alkylamino, such Z groups preferably having the number of carbon atoms as specified for corresponding X and Y groups; and
each N, Nxe2x80x2 and Nxe2x80x3 is the same or different and is a nucleoside group, particularly a nucleoside group capable of exhibiting antiviral activity. Preferred nucleoside groups are those that in the triphosphate form are capable of selectively inhibiting a viral DNA or RNA polymerase, i.e. inhibiting a viral DNA or RNA polymerase selectively with respect to host cell polymerases. Suitable nucleoside groups include substituted or unsubstituted purine or pyrimidine bases typically containing a sugar group or a sugar derivative (e.g. a sugar derivative suitably may be an acyclic nucleoside side chain that contains one or more of each of hydroxy and/or alkoxy groups and about 2 to 10 carbons, more typically about 3 to 8 carbons, such as the xe2x80x94CH2OCH2CH2OH side chain of acyclovir or the xe2x80x94CH2OCH(CH2OH)(CH2OH) side chain of ganciclovir). Generally preferred nucleoside N groups include 2xe2x80x2,3xe2x80x2-dideoxynucleosides or dioxanyl or thioxanyl analogues thereof, including those containing a purine or a pyrimidine base such as substituted and unsubstituted adenine, guanine, inosine, uracil, thymine, cytosine, etc. It also will be understood that in the above Formula I, the designation of xe2x80x9c(R, N or Z)xe2x80x9d, xe2x80x9c(Rxe2x80x2, Nxe2x80x2 or Zxe2x80x2)xe2x80x9d or xe2x80x9c(Rxe2x80x3, Nxe2x80x3 or Zxe2x80x3)xe2x80x9d indicates that one of R, N or Z, or Rxe2x80x2, Nxe2x80x2 or Zxe2x80x2 or Rxe2x80x3, Nxe2x80x3 or Zxe2x80x3 respectively is present at the specified ester position. N, Nxe2x80x2 and Nxe2x80x3 nucleoside groups may be employed in racemic or optically active form, and the anomeric carbon may have either the xcex1 or xcex2 configuration.
Preferred compounds of Formula I include phosphonacid/nucleoside conjugates where the carboxyl group of phosphonoformic acid or phosphonoacetic acid is linked via a C-ester bond to a lipophilic group, and the phosphonyl moiety is linked via a P-ester bond to a nucleoside group. More specifically, preferred are compounds of the following Formula II: 
wherein R, X, Y, W, Wxe2x80x2, m, Z and N are as defined above for Formula I.
Generally preferred compounds of Formula II include those where m is 0, i.e. compounds of the following Formula IIA: 
wherein R, W, Wxe2x80x2, Z and N are the same as defined above for Formula I.
Also preferred compounds of Formula I are compounds containing two nucleoside groups, including bis-nucleoside compounds of the following Formula III: 
wherein R, W, Wxe2x80x2, X, Y and m are the same as defined above for Formula I; and N and Nxe2x80x2 are the same or different and are each a nucleoside as defined above for N of Formula I.
Generally preferred compounds of Formula III include conjugates of phosphonoformic acid derivatives, i.e. compounds of the following Formula IIIA: 
wherein R, W, Wxe2x80x2, N and Nxe2x80x2 are the same as defined for Formula III above.
Still further, also preferred are compounds of Formula I where the phosphonyl moiety is linked via a P-ester bond to a lipophilic group and either one or both of the carboxyl group and phosphonyl moiety of phophonoformic acid or phosphonoacetic acid is linked to a nucleoside group. In particular, compounds of the following Formula IV are provided: 
wherein N, Nxe2x80x2 W, Wxe2x80x2, X, Y, R, Z and m are the same as defined above for Formula I.
Generally preferred compounds of Formula IV include conjugates of phosphonoformic acid derivatives, i.e. compounds of the following Formula IVA: 
wherein N, Nxe2x80x2 W, Wxe2x80x2, R and Z are the same as defined above for IV. It is understood that in the above Formulae IV and IVA the designation of xe2x80x9c(Nxe2x80x2 or Z)xe2x80x9d indicates that one of Nxe2x80x2 and Z is present at the P-ester position.
Also preferred compounds of Formula I are the following compounds of Formula V: 
wherein Z, W, Wxe2x80x2 X, Y, R, N and m are the same as defined for Formula I.
Again, generally preferred compounds of Formula V include conjugates of phosphonoformic acid derivatives, i.e. compounds of the following Formula VA: 
wherein Z, W, Wxe2x80x2, R, and N are the same as defined for Formula I.
As mentioned above, therapeutic compounds of the invention (i.e. compounds of Formulae I, IA, II, IIA, III, IIIA, IV, IVA, V and VA) are useful for treatment of viral infections, especially retroviral infections and in particular HIV infections, including treatment against HIV strains that exhibit resistance to current therapies. Compounds of the invention have been found to inhibit HIV-1 replication in cells infected with AZT-resistant HIV-1 strains as well as cells infected with PFA-resistant HIV-1 strains. Particularly preferred compounds of the invention exhibit EC50 values of about 10 xcexcM or less, and more preferably about 1 xcexcM or less against AZT resistant HIV-1 strains (such as A018-post) and/or Foscarnet resistant HIV-1 strains (such as LAI-E89K) in standard HIV plaque reduction assays, specifically the HIV Plaque Reduction Assay of the protocol specified in Example 27 which follows. References herein to xe2x80x9cHIV Plaque Reduction Assayxe2x80x9d are intended to refer to the protocol of that Example 27.
The invention further provides pharmaceutical compositions that comprise one or more compounds of the invention and a suitable carrier. In a particularly preferred aspect, compounds of the invention are formulated as liposomes. The invention also provides compounds useful to prepare compounds of the invention. Other aspects of the invention are disclosed infra.
We have now discovered that compounds of the following Formulae I, II, III, IV and V can be used to treat viral infections, particularly virally infected human cells, including cells infected with a retrovirus such as HIV, and thus the compounds can be used for treatment of HIV infected individuals: 
wherein R, Rxe2x80x2, Rxe2x80x3, W, Wxe2x80x2 X, Y, N, Nxe2x80x2, Nxe2x80x3, Z, Zxe2x80x2, Zxe2x80x3 and m are as defined above.
Optionally substituted alkyl, alkenyl, alkynyl, alkoxy and alkylthio, particularly non-cyclic alkyl, alkenyl, alkynyl, alkoxy and alkylthio, are generally preferred R groups of compounds of Formulae I, II, III, IV and V. Particularly suitable R groups include straight and branched chain alkyl, alkenyl, alkynyl, alkoxy and alkylthio optionally substituted by halogen, hydroxy and alkanoyl. Also suitable are R groups that contain one or more units of the following formula (A):
xe2x80x94(U)axe2x80x94(CR1R2)bxe2x80x94(U)cxe2x80x94xe2x80x83xe2x80x83(A)
wherein each U is independently a sulfur, oxygen, optionally substituted nitrogen, sulfinyl (xe2x80x94SOxe2x80x94), or sulfonyl (xe2x80x94SO2xe2x80x94);
R1 and R2 are each independently a hydrogen; halogen; nitro; optionally substituted alkyl having 1 to about 24 carbon atoms, more typically 1 to about 12 carbons; optionally substituted alkenyl having 2 to about 24 carbon atoms, more typically 2 to about 12 carbons; optionally substituted alkynyl having 2 to about 24 carbon atoms, more typically 2 to about 12 carbons; optionally substituted alkoxy having 1 to about 24 carbon atoms, more typically 1 to about 12 carbons; optionally substituted alkylamino having 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms; or optionally substituted alkylthio having 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms, or R2 is a carbon atom and R1 is a double or triple carbon-carbon bond to provide an alkenylene or alkynylene unit;
a is 0 or 1; b is 1 to about 30; and c is 0 or 1.
Particularly preferred R groups have the following formula (B): 
wherein R1, R1xe2x80x2 and R1xe2x80x3 are each independently optionally substituted alkoxy group preferably having from 1 to about 24 carbon atoms; optionally substituted alkylthio group preferably having from 1 to about 24 carbon atoms; optionally substituted alkylsulfinyl group preferably having from 1 to about 24 carbon atoms; optionally substituted alkylsulfonyl group preferably having from 1 to about 24 carbon atoms; or optionally substituted alkanoyl preferably having from 1 to about 24 carbon atoms;
each k is independently 0 or 1;
each R2 or R2 is independently hydrogen, xe2x95x90O, halogen, nitro, amino, methoxy, methylthio, xe2x80x94O-benzyl, xe2x80x94S-benzyl, amino substituted by alkanoyl having 1 to 24 carbon atoms and 0 to 3 double bonds, optionally substituted aminoalkyl having from 1 to 24 carbons and from 0 to 6 double bonds; and
each M is independently xe2x80x94C(R1)(R2)xe2x80x94 (wherein R1 and R2 are as defined in this formula (B) above) N, O, S, sulfinyl (SO) or sulfonyl (SO2);
n is an integer of from 0 to 6;
R3 and R3xe2x80x2 are each independently hydrogen; halogen; hydroxyl; nitro; sulfhydryl; amino; optionally substituted alkyl preferably having 1 to about 24 carbon atoms; optionally substituted alkenyl preferably having 2 to about 24 carbon atoms; optionally substituted alkynyl preferably having 2 to about 24 carbon atoms; optionally substituted alkoxy preferably having 1 to about 24 carbon atoms; optionally substituted alkylthio preferably, having 1 to about 24 carbon atoms; optionally substituted alkylsulfinyl preferably having 1 to about 24 carbon atoms; optionally substituted alkylsulfonyl preferably having 1 to about 24 carbon atoms; or optionally substituted alkylamino preferably having 1 to about 24 carbon atoms. (It is understood that the carbon of formulae (B) with R3 and R3xe2x80x2is directly bonded to the conjugate molecule.)
Additional preferred R groups of compounds of the invention have the following formula (C): 
wherein R1, R1xe2x80x2, R1xe2x80x3, k, R3 and R3xe2x80x2 are each the same as defined above for formula (B). (It is understood that the carbon of formulae (C) with R3 and R3xe2x80x2 is directly bonded to the conjugate molecule.)
Particularly preferred nucleosides groups (i.e. N, Nxe2x80x2or Nxe2x80x3 groups) of compounds of the invention include 2xe2x80x2,3xe2x80x2-dideoxynucleosides, especially 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT), and other 2xe2x80x2,3xe2x80x2-dideoxynucleosides such as 2xe2x80x2,3xe2x80x2-dideoxyinosine (ddI), 2xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxyinosine (F-ddI), 2xe2x80x2,3xe2x80x2-dideoxycytidine (ddC), 2xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxyadenosine (F-ddA), 5-fluoro-3xe2x80x2-thia-2xe2x80x2,3xe2x80x2-dideoxycytidine, trifluridine (Merck Index, 11th edition, 9599), d4T (stavudine), 3TC (lamivudine), vidarabine (Merck Index, 11th edition, 9881), idoxuridine (Merck Index, 11th edition, 4819), (xe2x88x92)-fialuridine ((xe2x88x92)-FIAU; (xe2x88x92)-1xe2x80x2,2xe2x80x2-deoxy-2xe2x80x2-fluoro-1-xcex2-D-arabinofuranosyl-5-iodouracil), d4T, 3TC, 1592U89, sorivudine (BV-araU), (+), (xe2x88x92) or (xc2x1) FTC ((+), (xe2x88x92) or (xc2x1) 5-fluoro-1-2-(hydroxymethyl)-(1,3-oxathiolan-5-yl)cytosine), xcex2-D-2,6-diaminopurine-dioxolanyl (DAPD), and 2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-azido-5-methylcytidine (CS92). Acyclovir, ganciclovir and penciclovir ((9-[4-hydroxy-3-(hydroxymethylbut-1-yl]guanine) are also preferred nucleoside groups of compounds of the invention. A nuceloside group (N, Nxe2x80x2 or Nxe2x80x3) is suitably covalently linked to a conjugate of the invention at the site of a hydroxyl group of a sugar or sugar derivative of the nucleoside group (e.g. as exemplified in the Schemes which follow), although other linkages at other positions of a nucleoside group also will be suitable.
Preferred Z groups of compounds of Formulae I, II, III, IV and V include physiologically acceptable cations such as an ammonium or quaternary ammonium cation. Oxygen is a typically preferred W and/or Wxe2x80x2 group of compounds of Formulae I, II, III, IV and V. Compounds where at least one of W and Wxe2x80x2 is sulfur also will be preferred.
Suitable halogen substituent groups of compounds of the invention are F, Cl, Br and I. As used herein, the term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although cyclic groups will comprise at least three carbon ring atoms. Alicyclic alkyl groups are generally preferred. Alkenyl and alkynyl groups of compounds of the invention have one or more unsaturated linkages, typically 1 to about 3 or 4 unsaturated linkages. Also, the terms alkenyl and alkynyl as used herein refer to both cyclic and noncyclic groups, although straight or branched noncyclic groups are generally more preferred. Alkoxy groups of compounds of the invention have one or more oxygen linkages, typically 1 to about 5 or 6 oxygen linkages. Alkylthio groups of compounds of the invention have one or more thioether linkages, typically 1 to about 5 or 6 thioether linkages. Alkylsulfinyl groups of compound of the invention have one or more sulfinyl (SO) linkages, typically 1 to about 5 or 6 sulfinyl linkages. Alkylsulfonyl groups of compounds of the invention have one or more sulfonyl (SO2) linkages, typically 1 to about 5 or 6 sulfonyl linkages. Preferred alkylamino groups of compounds of the invention include those groups having one or more primary, secondary and/or tertiary amine groups, preferably 1 to about 3 or 4 amine groups. Suitable alkanoyl groups have one or more carbonyl groups, typically 1 to about 4 or 5 carbonyl groups. Alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkanoyl and other groups may be suitably either linear or branched.
As discussed above, R, Rxe2x80x2, Rxe2x80x3 (including R, Rxe2x80x2, Rxe2x80x3 groups of formulae (A), (B) and (C)), X, Y, Z, nucleoside and other groups are optionally substituted. Suitable groups that may be present on a xe2x80x9csubstitutedxe2x80x9d R, Rxe2x80x2, Rxe2x80x3, X, Y, Z, nucleoside or other substituent include e.g. halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; nitro; azido; sulfhydryl; alkanoyl e.g. C1-6 alkanoyl group such as acetyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon atoms, preferably from 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkylsulfinyl groups including those moieties having one or more sulfonyl linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkylsulfonyl groups including those moieties having one or more sulfonyl linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; aminoalkyl groups such as groups having one or more N atoms and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; carbocyclic aryl having 6 or more carbons, particularly phenyl; aryloxy such as phenoxy; aralkyl having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with benzyl being a preferred group; aralkoxy having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with O-benzyl being a preferred group; or a heteroaromatic or heteroalicyclic group having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl. A xe2x80x9csubstitutedxe2x80x9d R, Rxe2x80x2 Rxe2x80x3, X, Y, Z, nucleoside or other substituent of a compound of the invention may be substituted at one or more available positions, typically 1 to about 3 positions, by one or more suitable groups such as those listed immediately above.
It was also indicated that in Formula (A), U may be substituted to provide a tertiary amine. Such a substituted nitrogen may be substituted by suitable groups set forth above with respect to other substituted groups, particularly substitutions of alkyl, alkoxy, alkylthio and aminoalkyl groups.
Specifically preferred compounds of the invention include:
3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-(1-octadecyloxycarbonyloxyphosphinyl)thymidine;
3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-(1-eicosanyloxycarbonyloxyphosphinyl)thymidine;
3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-(1-docosanyloxycarbonyloxyphosphinyl)thymidine;
3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-[(3xcex2-cholest-5-enyl)oxycarbonyloxyphosphinyl)]thymidine;
di-O-(3xe2x80x2-azido-3xe2x80x2-deoxythimidin-5xe2x80x2-yl)-1-octadecyloxycarbonylphosphonate;
di-O-(3xe2x80x2-azido-3xe2x80x2-deoxythimidin-5xe2x80x2-yl)-1-eicosanyloxycarbonylphosphonate;
di-O-(3xe2x80x2-azido-3xe2x80x2-deoxythimidin-5xe2x80x2-yl)-1-docosanyloxycarbonylphosphonate;
di-O-(3xe2x80x2-azido-3xe2x80x2-deoxythimidin-5xe2x80x2-yl)-3xcex2-cholest-5-enyloxycarbonylphosphonate; sodium 3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-(hexadecyloxypropoxy)carbonyloxyphosphinyl thymidine; and 3xe2x80x2-azido-3xe2x80x2-deoxy-5xe2x80x2-O-[(hexadecyloxypropoxy) (hydroxy)phosphono(carbonyl]thymidine.
In certain preferred aspects, the invention includes compounds of Formulae IIxe2x80x2 and IIAxe2x80x2, which are defined the same as above for Formulae II and IIA respectively, except that the group R does not contain heteroatoms (N, O, S), or at least the group R does not contain oxygen or thio substitution. Compounds of Formula IIxe2x80x2 and IIAxe2x80x2 can be used in the therapeutic methods disclosed herein including to treat viral infections, particularly cells infected with HIV and drug-resistant HIV strains.
As mentioned above, the invention also includes novel intermediate compounds useful to prepare compounds the invention. Specifically, chloroformates of the following Formula VI are provided: 
wherein R is as defined above in Formula I.
Additional intermediate compounds of the invention are carbonylphosphonic acids of the following Formula VII: 
wherein R is the same as defined above for Formula I; and R1 and R2 may be the same or different and are hydrogen; a counter cation e.g. a pharmaceutically acceptable cation such as an alkali metal or earth metal, e.g. sodium, potassium, lithium, etc., or an ammonium or quaternary ammonium cation (e.g. NZ4+ where Z is C1-4 alkyl); or alkyl, preferably having 1 to about 12 carbons, more preferably 1 to about 3 carbons.
Compounds of Formula VII also may be used in the therapeutic methods disclosed herein, including to treat viral infections, particularly cells infected with HIV.
Compounds of the invention can be prepared as generally depicted in the following Schemes I through VI. In the discussions of those Schemes, the group R is the same as defined above for Formulae I, II, III, IV and V. Additionally, for purposes of exemplification only, a preferred nucleoside (group N, Nxe2x80x2, Nxe2x80x3 in Formulae I through V) of AZT is depicted in the Schemes, and it will be understood that a wide variety of nucleosides can be employed in the same manner as discussed below for AZT. Similarly, compounds exemplified in the Schemes are phosphonoformic acid derivatives, i.e. compounds of Formulae I, II, III, IV and V (and corresponding intermediates) where m is 0, although other phosphonoacid derivatives can be similarly employed to provide compounds of Formulae I-V where m=1. Compounds of the invention where W and/or Wxe2x80x2 is sulfur or Se also can be prepared as shown in the following Schemes with substitution of appropriate starting materials, e.g. a thio or selenium reagent. See, for instance, WO 96/39831. 
As shown in Scheme I, for preparation of compounds of Formulae I, II and III of the invention, a carbonylphosphonate 4 is suitably prepared by reaction of the corresponding alcohol ROH with phosgene or triphosgene ((Cl3CO)2CO) to provide the chloroformate 3. See Examples 1-3 which follow for exemplary reaction conditions. Arbuzov reaction of 3 with a trialkyl phosphite such as trimethylphosphite yields the triester 4. See Examples 4-7 which follow for exemplary conditions.
Selective removal of one alkyl group by overnight treatment of the triester 4 with NaI in a suitable solvent such as dimethylformamide with or without tetrahydrofuran or acetone as a co-solvent provides the diester 5 in generally good yields with generally little or no purification required other than washing with hexane or other suitable solvent to remove any unreacted starting material. See Examples 8-11 which follow.
Treatment of triester 4 with Me3SiBr in a suitable solvent such as methylene chloride for a time and temperature sufficient for reaction completion (e.g. room temperature for about four hours) provides phosphonic acid 6. See Examples 12-15 which follow. The phosphonic acid 6 typically will be highly hygroscopic and should be stored appropriately, e.g. xe2x88x9220xc2x0 C. in a tightly sealed container. The acid 6 can be converted to the corresponding salt 7, e.g. by treatment with two molar equivalents of NaOMe in methanol. See Examples 16-19 which follow.
As depicted in Schemes II through IV below, compounds of Formulae I, II and III can be prepared by several routes from the above discussed intermediates. Thus, as shown in the following Scheme II, triester 4 is reacted with excess PCl5 in a suitable solvent and for a time and temperature sufficient to form monochloro intermediate 8, e.g. in refluxing CCl4 for three hours under an inert atmosphere such as dry argon. Excess PCl5 can be destroyed by bubbling SO2 gas through the reaction mixture. The resulting reaction mixture can be evaporated to dryness under reduced pressure, the residue of 8 taken up in a suitable solvent such as dry dimethylformamide and cooled to xe2x88x9250xc2x0 C. or other suitable temperature. The nucleoside reagent in moderate molar excess (e.g. 1.5 molar equivalents) is then added to the solution and the mixture stirred for a time and temperature sufficient for reaction completion, e.g. for 24 hours at room temperature. See Method A of Example 20 for an exemplary procedure. The resulting compound 9 can be purified e.g. by flash chromatography and optionally further reacted to provide other compounds of the invention. For example, the Pxe2x80x94OCH3 ester 9 shown in Scheme II below can be treated at room temperature for 24 hours with about 1.4 to 1.5 equivalents of NaI in tetrahydrofuran under argon in the absence of light followed by ion-chromatography on DEAE-cellulose with NH4HCO3 as the eluent to provide compound 10 where X is ONH4. 
Compounds of Formula III can be prepared in similar manner as shown in Scheme II above with modification of reaction conditions to favor formation of the bis-nucleoside conjugate. Thus, for example, as shown in Scheme III below, triester 4 is reacted with excess PCl5 in a suitable solvent and for a time and temperature sufficient to form the dichloro intermediate 11, for example in refluxing CCl4 for 48 hours under an inert atmosphere such as dry argon. Intermediate 11 is then reacted with a relatively large molar excess of one or more nucleoside reagents and for extended periods, e.g. with about 3 molar equivalents of AZT for 48 hours, to provide bis-nucleoside compound 12. See Example 24 for exemplary reaction conditions. 
An alternative route to compounds of Formulae I through III is generally shown in Scheme IV below. Intermediate 5 is reacted with an excess of the nucleoside reagent and Cl3CCN in pyridine for a time and temperature sufficient for reaction completion, e.g. reaction with excess AZT under argon at about 50 to 60xc2x0 C. overnight, and with the rigorous exclusion of moisture. See Method B of Example 20 for an exemplary procedure. The resulting compound 13 of Formula I can be further reacted to provide other compounds of the invention, e.g. flash chromatography on silica gel with 85:15:1 CHCl3xe2x80x94MeOH-28%NH4OH as the eluent to provide compound 10 (i.e. Xxe2x95x90ONH4). This Cl3CCN method is generally preferred as a more direct route that can provide higher overall yields than the above discussed PCl5 procedure. 
Compounds of the invention having structures of Formulae IV are suitably prepared as generally depicted in the following Schemes V and VI. 
Thus, as shown in Scheme V, carbonyl nucleoside ester 14 is prepared by reaction of a dialkylphosphonoformic chloride with a desired nucleoside, e.g. AZT as shown above, under suitable conditions such as addition of a pyridine solution of diethylphosphonoformic chloride to a solution of the nucleoside in pyridine suitably in the presence of a catalyst such as N,N-dimethylaminopyridine followed by overnight stirring of the reaction mixture. See Example 25, Part A for exemplary conditions. Treatment of triester 14 with Me3SiBr in a suitable solvent such as dry acetonitrile for a time and temperature sufficient for reaction completion provides the diacid 15 which can be isolated under reduced pressure and used for further reaction without additional purification. Thus, the isolated diacid 15 can be reacted with a lipophilic alcohol 16 to provide the P-ester 17 in the presence of a coupling agent. See, for instance, Example 25.
Scheme VI depicts a suitable preparation of additional compounds of Formula V. 
Thus, as shown in Scheme VI, lipophilic alcohol 16 is reacted with a dialkylphosphorochloridite in the presence of base to provide the lipophilic phosphite 18, which can be purified as desired e.g. by flash chromatography. The phosphite 18 is then reacted with an alkyl chloroformate to provide phosphonate 19. Treatment of the phosphonate 19 with a slight molar excess of PCl5 in a suitable solvent and for a time and temperature sufficient to form a reactive phosphonyl chloride intermediate. Suitable reaction conditions include refluxing the phosphonate 19 and PCl5 in CCl4 for about three hours or more. Excess PCl5 can be destroyed by bubbling SO2 gas through the reaction mixture. The phosphonyl chloride intermediate can be isolated under reduced pressure and the resulting residue taken up in dry dimethylformamide or other suitable solvent and cooled to xe2x88x9250xc2x0 C. or other suitable temperature. The nucleoside reagent is then added to the phosphonyl chloride solution and the resulting mixture stirred for a time and temperature sufficient for reaction completion, e.g. for about 24 hours at room temperature. The resulting triester conjugate 20 can be purified as desired, e.g. by flash chromatography. See Example 26, Part C for an exemplary procedure. The conjugate 20 is further reacted to provide compounds of the invention, e.g. the triester 20 can be hydrolyzed under basic conditions to provide a carboxy salt 21, i.e. where Nxe2x80x3 of 21 is a pharmaceutically acceptable cation. That salt or the chloroformate derivative thereof (i.e. the compound RO(NO)P(xe2x95x90O)C(xe2x95x90O)Cl) then can be reacted with a nucleoside reagent such as AZT to provide the bis-nucleoside compound 21 where Nxe2x80x2 is a nucleoside group.
As discussed above, the invention includes methods for synthesis of the compounds of the invention. Thus, the invention includes methods for preparation of compounds of Formula III which includes reacting a carbonylphosphonic acid or carbonylphosphonic acid halide such as an acid di-halide 11 shown in Scheme III above with a molar excess of a nucleoside, preferably about a two or 2.5 or more molar excess of a nucleoside at a time and temperature suitable for reaction. The invention also includes methods for preparation of compounds of Formulae II and III by reaction of a phosphonic acid substituted by a lipophilic R group (e.g. intermediate 5 of Scheme IV) with a nucleoside (preferably used in molar excess) and Cl3CCN, preferably in a suitable solvent such as a pyridine, for a time and temperature sufficient for reaction completion.
The invention thus provides methods of treatment against virus infections and diseases associated with viruses, which methods in general will comprise administration of a therapeutically effective amount of one or more compounds of Formulae I, II, III, IV or V to a mammal, particularly a human, suffering from or susceptible to a viral infection or disease otherwise associated with a virus.
Compounds of the invention will be useful to treat cells infected with a virus capable of causing an immunodeficiency disease, particularly in a human. Compounds of the invention will be particularly useful to treat retroviral infection in cells and in a human, particularly HIV infected human cells. Specific examples of retroviral infections which may be treated in accordance with the invention include human retroviral infections such as HIV-1, HIV-2, and Human T-cell Lymphotropic Virus (HTLV) e.g. HTLV-I or HTLV-II infections.
The invention also provides methods of treatment of other diseases caused by or otherwise associated with a virus such as influenza including influenza A and B as well as diseases associated with viruses of the herpes family, e.g., herpes simplex viruses (HSV) including herpes simplex 1 and 2 viruses (HSV 1, HSV 2), varicella zoster virus (VZV; shingles), human herpes virus 6, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and other herpes virus infections such as feline herpes virus infections, and diseases associated with hepatitis viruses including hepatitis B viruses (HBV) B virus. Examples of clinical conditions which are caused by such viruses include herpetic keratitis, herpetic encephalitis, cold sores and genital infections (caused by herpes simplex), chicken pox and shingles (caused by varicella zoster) and CMV-pneumonia and retinitis, particularly in immunocompromised patients including renal and bone marrow transplant patients and patients with Acquired Immune Deficiency Syndrome (AIDS). Epstein-Barr virus can cause infectious mononucleosis, and is also suggested as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma and Burkitt""s lymphoma.
As discussed above, particularly preferred compounds of the invention are active against drug-resistant viral strains, and it has been surprisingly found that compounds of the invention are highly active against HIV strains that are PFA-resistant as well as HIV strains that are AZT-resistant.
Without wishing to be bound by theory, it is believed the multiple and distinct covalently linked antiviral agents (i.e. a nucleoside and a phosphonoacid) of compounds of the invention make it more difficult for a virus to successfully mutate to any one of the linked agents.
Moreover, by virtue of the covalent linkage, the conjugates of the invention present the nucleoside and phosphonacid compounds to a virus essentially simultaneously, an effect that may not be readily achieved by administering the same compounds in a drug xe2x80x9ccocktailxe2x80x9d formulation without covalently linking the compounds.
It also has been reported that treatment with a 2xe2x80x2,3xe2x80x2-dideoxynucleoside such as AZT can sensitize a patient to Foscarnet, and treatment with Foscarnet can in turn sensitize a patient to a 2xe2x80x2,3xe2x80x2-dideoxynucleoside such as AZT. See G. Tachedjian et al., Virology, 212:58-62 (1995); and G. Tachedjian et al., Virology, 70:7171-7181 (1996). Accordingly, the essentially simultaneous presentation to a virally infected cell of a nucleoside and phosphonacid via a conjugate of the invention may provide synergistic results, as is indicated by the data shown in the examples which follow, including Example 28.
Administration of compounds of the invention may be made by a variety of suitable routes including oral, topical (including transdermal, buccal or sublingal), nasal and parenteral (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection) with oral or parenteral being generally preferred. It also will be appreciated that the preferred method of administration and dosage amount may vary with, for example, the condition and age of the recipient.
Compounds of the invention may be used in therapy in conjunction with other medicaments such as reverse transcriptase inhibitors such as a dideoxynucleoside including AZT, ddI, ddC, d4T, 3TC, FTC, DAPD, 1592U89 or CS92; TAT antagonists such as Ro 3-3335 and Ro 24-7429; protease inhibitors such as saquinavir, ritonavir, indinavir or AG1343 (Viracept); and other agents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), ganciclovir or penciclovir, interferon, e.g., alpha-interon or interleukin II, or in conjunction with other immune modulation agents including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate.
While one or more compounds of the invention may be administered alone, they also may be present as part of a pharmaceutical composition in mixture with conventional excipient, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, oral or other desired administration and which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
For parenteral application, particularly suitable are solutions, preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories. Ampules are convenient unit dosages.
For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch. A syrup, elixir or the like can be used wherein a sweetened vehicle is employed. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
Therapeutic compounds of the invention also may be incorporated into liposomes. The incorporation can be carried out according to known liposome preparation procedures, e.g. sonication and extrusion. Suitable conventional methods of liposome preparation are also disclosed in e.g. A. D. Bangham et al., J. Mol. Biol., 23:238-252 (1965); F. Olson et al., Biochim. Biophys. Acta, 557:9-23 (1979); F. Szoka et al., Proc. Nat. Acad. Sci., 75:4194-4198 (1978); S. Kim et al., Biochim. Biophys. Acta, 728:339-348 (1983); and Mayer et al., Biochim. Biophys. Acta, 858:161-168 (1986).
The liposome may be made from one or more of the conjugates of Formulae I-V alone, or more preferably, in combination with any of the conventional synthetic or natural phospholipid liposome materials including phospholipids from natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine or phosphatidylinositol. Synthetic phospholipids also may be used e.g, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidycholine and corresponding synthetic phosphatidylethanolamines and phosphatidylglycerols. Cholesterol or other sterols, cholesterol hemisuccinate, glycolipids, 1,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP), N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and other cationic lipids may be incorporated into the liposomes. The relative amounts of the one or more compounds of Formulae I-V and additives used in the liposomes may vary relatively widely. Liposomes of the invention suitably contain about 60 to 90 mole percent of natural or synthetic phospholipid; cholesterol, cholesterol hemisuccinate, fatty acids or cationic lipids may be used in amounts ranging from 0 to 50 mole percent; and the one or more therapeutic compounds of the invention may be suitably present in amounts of from about 0.01 to about 50 mole percent.
Additionally, the lipophilic R groups of compounds of the invention can enable preparation of liposomes where compound(s) of Formulae I-V are substantially incorporated into the lipid bilayer of a liposome and the aqueous liposome compartment may contain one or more other drugs such as an antiviral nucleoside (preferably AZT, ddI, ddC, d4T, 3TC or 1592U89) or other of the antiviral agents discussed above to provide an effective xe2x80x9ccocktailxe2x80x9d formulation system.
It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.
In general, for treatment of immunodeficiency infections, particularly an HIV infection, a suitable effective dose of one or more compounds of Formulae I, II, III, IV or V will be in the range of from 0.01 to 100 milligrams per kilogram of bodyweight of recipient per day, preferably in the range of from 0.1 to 50 milligrams per kilogram bodyweight of recipient per day, more preferably in the range of 1 to 20 milligrams per kilogram bodyweight of recipient per day. The desired dose is suitably administered once daily, or several sub-doses, e.g. 2 to 5 sub-doses, are administered at appropriate intervals through the day, or other appropriate schedule.
All documents mentioned herein are incorporated herein by reference.
The present invention is further illustrated by the following examples. These examples are provided to aid in the understanding of the invention and are not to be construed as limitations thereof.
In the following examples 1-24, IR spectra were obtained on a Perkin-Elmer Model 781 double-beam recording spectrophotometer; 1H NMR spectra were obtained on Varian Model EM360L and Bruker AM-500 instruments at 60 and 500 MHz, respectively, with Me4Si as the reference. TLC was performed on Whatman MK6F and Baker 250F silica gel plates with a fluorescent indicator dye. Spots were visualized under a 254-nm UV lamp, in an iodine chamber, or by spraying with H2SO4/H2O/EtOH or molybdic acid spray reagent. Melting points were obtained on a Fisher-Johns hot-stage apparatus and are not corrected. Chemicals were purchased from Aldrich (Milwaukee, Wis.), Sigma (St. Louis, Mo.), and Fisher (Boston, Mass.). Solvents were routinely stored over Linde 4xc3x85 molecular sieves. Microchemical analyses were done by Quantitative Technologies, Inc., Whitehouse, N.J.